STRUCTURE AND FUNCTION OF RESTRICTION ENDONUCLEASES

结合了序列特异性和催化化学的元素是 开发针对特定靶点的化疗药物的中心问题 核酸序列和结构。限制性内切酶，其中EcoRI 核酸内切酶是最具代表性的，是最简单的生物制剂 特定部位的核酸化学，但这其中的许多机械方面 水解性仍未确定。这个项目采用了一种独特的 用于：i)鉴定蛋白质和底物的技术组合 所需金属离子的配体(MG(II))，并定义 反应中的这些相互作用。这些目标将通过以下方式实现 定点突变，金属离子替代，底物类似物， 惰性过渡金属络合物和磁共振波谱。Ii) 用光谱学表征所需金属离子的水化 方法和探讨质子转移在DNA水解中的重要性 利用动力学同位素效应。三)探索构象的作用 介导EcoRI内切酶底物特异性和STAR的变化 活动。用3-氟-酪氨酸标记的EcoRI内切酶将被 准备好了并进行了表征。19/F核磁共振波谱将应用于此 确定底物特异性和StAR活性的酶衍生物 在构象上有物质基础。从这些网站获得的信息 实验将确定酶结构和酶之间的关系， 所需的金属离子，以及衬底以及澄清工作 EcoRI活性的模型。了解结构和功能 这种特定的自然形式的水解性催化是一种可行的 最终设计特定于序列的剪刀的策略 针对与癌症和艾滋病相关的特定基因。

Combining elements of sequence-specificity and catalytic chemistry is the central problem in developing chemotherapeutics which target specific nucleic acid sequences and structures. Restriction enzymes, of which EcoRI endonuclease is most representative, are the simplest biological agents of site-specific nucleic acid chemistry, yet many mechanistic aspects of this hydrolytic activity remain undefined. This project employs a unique combination of techniques to: i) Identify the protein and substrate ligands of the required metal ion (MG(II)) and define the importance of these interactions in the reaction. These goals will be accomplished with site-directed mutagenesis, metal ion substitution, substrate analogs, inert transition metal complexes, and magnetic resonance spectroscopy. ii) Characterize the hydration of the required metal ion by spectroscopic methods and probe the importance of proton transfer in DNA hydrolysis using kinetic isotope effects. iii) Probe the role of conformational changes in mediating EcoRI endonuclease substrate specificity and star activity. EcoRI endonuclease labelled with 3-fluoro--tyrosine will be prepared and characterized. 19/F NMR spectroscopy will be applied to this enzyme derivative to determine if substrate specificity and star activity have a physical basis in conformation. Information obtained from these experiments will identify the relationships between the enzyme structure, the required metal ion, and the substrate as well as clarify working models of EcoRI activity. Understanding the structural and functional aspects of this natural form of specific, hydrolytic catalysis is a viable strategy to the eventual design of sequence-specific scissors which can target specific genes linked to cancer and AIDS.